Electron tomography (ET) is an important tool for determining three-dimensional subcellular structures. We have implemented ET in a 300 kV transmission electron microscope to determine the three-dimensional organization of supramolecular assemblies in a variety of biological systems ranging from simple cytoskeletons in bacteria to large protein complexes in neurons. It is not always feasible to obtain cryo-electron tomographic data from specimens maintained in their frozen hydrated state. In those cases, useful results can often be obtained by rapidly freezing the cells, freeze-substituting the water for solvent, embedding in plastic and sectioning at room temperature. We have collected dual axis tilt series from such freeze-substituted specimens and performed three-dimensional reconstructions using the IMOD program (University of Colorado). An advantage of this approach is that dual-axis tilt series can be acquired more easily, which reduces artifacts due to the missing wedge in the reconstruction. To gain insight into the structural basis of motility in the simplest free-living cell, the wall-less bacterium Spiroplasma, we have obtained tomographic reconstructions of Spiroplasma's cytoskeletal ribbon. We find that the cytoskeleton is composed of a major protein Fib anchored to the underlying membrane by another protein that we identify as the ATPase MreB, a bacterial analog of actin. We have also determined the number of ribosomes per unit volume of Spiroplasma and have identified a network of filaments as the cell's DNA. Electron tomography has also been applied to elucidate the structure of a highly complex supramolecular assembly, the post-synaptic density (PSD), which could eventually lead to a better understanding of neurological diseases. The PSD, which is embedded in the postsynaptic membrane, contains receptors, scaffold molecules, and cytoskeletal elements and is the primary postsynaptic site for signal transduction and signal processing. The PSDs at excitatory synapses contain glutamate receptors of the NMDA and AMPA type. Recycling of AMPA receptors at the PSD accounts for dynamic changes in synaptic transmission. The PSD is known to contain hundreds of different proteins and has been extremely difficult to study by conventional structural techniques. Our electron tomograms recorded from suitably stained freeze-substituted neurons of cultured rat brain showed that PSDs contain vertically oriented filaments, which intertwine with horizontally oriented filaments lying close to the postsynaptic membrane, and define an orthogonal interlinked scaffold at the core of the PSD. The thicket of vertical filaments gives rise to the typical dense appearance that is characteristic of PSDs in standard EM cross-sectional views. Vertical filaments are ubiquitous in reconstructions of PSDs, even in places where other structural elements are absent. Based on the observed number per unit area, it is estimated that there are approximatley 400 vertical filaments in a 400-nm-diameter PSD. The dimensions of vertical filaments, and their associations with the postsynaptic membrane, suggest that they belong to the PSD-95 family of MAGUK proteins. Vertical filaments contact two types of transmembrane structures whose sizes and positions match those of glutamate receptors and intermesh with two types of horizontally oriented filaments lying 10 to 20 nm from the postsynaptic membrane. The longer horizontal filaments link adjacent NMDAR-type structures, whereas the smaller filaments link both NMDA- and AMPAR-type structures. The orthogonal, interlinked scaffold of filaments at the core of the PSD provides a structural basis for understanding dynamic aspects of postsynaptic function. Immuno-electron microscopy helps to identify specific proteins like PSD-95 within the postsynaptic density complex. Filaments segmented in a series of virtual sections were classified on the basis of their location, shape, and dimensions. A large class of membrane-associated filaments at the PSD is nearly straight and vertically oriented with respect to the postsynaptic membrane. We refer to filaments of this type as vertical filaments. Vertical filaments are typically 5 nm in diameter and 20 nm long. Vertical filaments within the PSD are uniformly spaced, with a nearest neighbor distance of 13 nm. Our results have demonstrated that ET combined with automated data acquisition in a 300 kV TEM provides useful 3-D structural information about the organization of large protein assemblies in a wide variety of cell types that are prepared by rapid freezing and freeze-substitution.